Although tubulin glutamylation is dysregulated in human ciliopathies, the in vivo function of this post- translational modification is not well understood. This gap in our knowledge represents a significant problem in understanding the underlying causes of ciliary dysfunction. The long-term goal is to elucidate the molecular mechanisms by which tubulin glutamylation regulates microtubule function to impact organismal development using C. elegans, an established model for the study of ciliary and cytoplasmic microtubules. The overall objectives of this proposal are to identify mechanisms by which glutamylation influences the function of microtubules both in non-motile cilia and more generally in the cytoplasm, and to elucidate the redundancies between all glutamylating (TTLL) enzymes. Our central hypothesis is that tubulin glutamylation is required for correct microtubule function, but due to overlap in enzymatic activity and expression, redundancy between the TTLL enzymes masks the function of tubulin glutamylation, an idea supported by preliminary data produced in the applicant's laboratory. The rationale for the proposed research is that by elucidating how glutamylation contributes to cilia and microtubule function, we will be able to determine critical factors underlying ciliopathies, aiding the development of treatments for these currently intractable diseases that affect close to 1 in 1000 individuals. The hypothesis will be tested by pursuing three specific aims: 1) Determine the function of the glutamylation-initiating TTLL enzymes in cilia; 2) Identify a mechanism by which glutamylation influences global (non-cilia) microtubule function; and 3) Identify further redundancies between all combinations of TTLL enzymes. Preliminary data show that combining mutations in the C. elegans TTLL enzymes reveals new phenotypes, indicating the existence of redundancy. In aim one a microscopic analysis of phenotypes associated with loss of three glutamylation-initiating enzymes will aid understanding of the function of glutamylation in non-motile cilia. The relationship between glutamylation and enzyme-mediated severing of cytoplasmic microtubules will be explored in aim 2, using glutamylation deficient worms. In order to identify further redundancies (aim 3) all combinations of mutations in TTLL enzymes will be constructed and phenotyped. The approach is innovative because by undertaking a genetic analysis of the glutamylating enzymes in C. elegans, we circumvent two major hurdles. First, most organisms have large numbers of TTLL enzymes but the worm possesses only five, making combinatorial analysis feasible. Second, C. elegans lacks a competing modification, glycylation, which complicates analyses in other organisms. Notably, this analysis uses an intact organism allowing exploration of the complexities of glutamylation in a physiologically relevant context. The proposed research will be significant as it is expected to advance our understanding of how tubulin glutamylation impacts cilia function and therefore how dysregulation contributes to human disease. Ultimately such knowledge can guide development of new treatment strategies.